Nucleoside derivatives of 5-methylcytosine suppress 5-azacytidine-induced reactivation of a silent transgene in suspension-cultured tobacco cells

Epigenetic modifications, including DNA methylation, are involved in the regulatory mechanisms of gene expression in animals and plants. In this study, we investigated whether the action of 5-azacytidine (5-aza-Cd), which is a well-known DNA methylation inhibitor, in suspension-cultured tobacco cells is affected by treatment with nucleoside derivatives of 5-methylcytosine (5-mCs), namely 5-methylcytidine (5-mCd) and 5-methyl-2′-deoxycytidine (5-mdCd). In a tobacco cell line, 5-aza-Cd treatment reactivated an epigenetically silenced transgene containing the cauliflower mosaic virus 35S promoter fused to the β-glucuronidase coding region and the nopaline synthase polyadenylation signal. The reactivation was evident on the fifth day of treatment and was augmented during culture with application of 5-aza-Cd at every subcultivation. This treatment, provided only once in the initial culture, resulted in transient transgene reactivation, followed by attenuation of its activity. The reactivation induced by 5-aza-Cd was suppressed by concomitant treatment with either 5-mCd or 5-mdCd. These results suggest that the 5-mCs derivatives inhibit and/or reverse 5-aza-Cd-induced reactivation of a silent transgene in tobacco cells.